Project Summary Mosquito-borne members of the Flavivirus family including Zika virus (ZKV), Dengue virus (DNV) and West Nile virus (WNV), are classified as re-emerging pathogens due to the frequency and severity of recent epidemics. Also known as arboviruses, these viruses are the etiologic agents of many debilitating diseases affecting the human population worldwide. Consequently, vector borne diseases now account for 17% of all infections worldwide. DNV is the fastest growing arboviral disease currently affecting 400 million annually with 96 million cases manifesting into clinical severity and 22,000 deaths, mainly children. WNV is considered the most important causative agent of viral encephalitis worldwide. The recent ZKV infection outbreak has been associated with congenital microcephaly and intracranial calcification and, in adults, with GBS and severe thrombocytopenia. Currently there is no effective treatment for infections caused by these viruses, which highlights the urgent need to find preventive and therapeutic interventions. The Flaviviridae genome is translated into a single polyprotein which is processed to yield 3 structural and 7 nonstructural proteins. The correct processing of the polyprotein is essential for replication of all flaviviruses, which requires both host proteases and the highly conserved viral NS2B-NS3 protease (NS2B-NS3pro). Hence, the viral protease is a rational target for development of small molecule inhibitors that block flavivirus replication. Small molecule antivirals targeting HIV-1-encoded and HCV-encoded proteases have been successfully developed, which supports the concept of developing chemotherapeutic agents targeting the flavivirus NS2B-NS3pro. The innovation of our proposal is: (i) optimization of a highly-sensitive screening assay for the identification of low binding fragment hits; (ii) evolution of fragment and compound hits into broad-spectrum leads; (iii) the use of replicon and plaque assays to test for cellular efficacy and guide optimization. Our preliminary results and the use of multiple cell-based models supports the feasibility of the discovery of broad-spectrum anti-flaviviral therapeutics. The specific aims are: Aim 1: Complete the screening of fragment and compound libraries for the identification of broad-spectrum NS2B-NS3pro hits. Milestone 1: Identify 6-8 structurally distinct broad-spectrum NS2B-NS3pro hits with an IC50 ? 25?M. Aim 2a: Iterative 3D-structure and SAR-based discovery of three non-overlapping broad-spectrum NS2B-NS3pro inhibitor series, using a combination of: (i) commercial analogues and (ii) med-chem design and synthesis approach. Milestone 2: Identify three non-overlapping broad-spectrum NS2B- NS3pro inhibitor series with an IC50 ? 200nM. Aim 2b: Characterize biochemically potent inhibitors for: (a) mode of inhibition and (b) enzyme:ligand interactions and prioritize compounds with IC50 ? 200nM, for cellular efficacy studies. Aim 3a: In vitro evaluation and optimization of biochemically potent compounds for cellular efficacy (EC50) and cytotoxicity (CC50). Milestone 3: Identify 4-6 lead compounds from each series with EC50 ? 5?M and CC50 ? 200?M. Aim 3b: Conduct in vitro ADME-based lead optimization of compounds with broad spectrum in vitro activity and acceptable therapeutic indices. Phase I Milestone: Identify 2-4 lead compounds exhibiting high bioavailability, weak inhibitor of CYPP450s, optimum stability and are not hERG channel blockers.